Nucleic acids encoding chimeric proteins comprising BMP-2 and a proteinase inhibitor

ABSTRACT

This invention relates to an agent for producing a pharmaceutical drug for postoperative use after removal of bone tumors produced from a nucleic acid by linking a known sequence for promoting bone growth and a known proteinase inhibitor by a variable spacer molecule. This linkage results in a novel bifunctional active ingredient combining both properties in a biological molecule. This invention is used in the medical field, in particular in the specialty field of orthopedics.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. §371 National Phase Entry Applicationfrom PCT/DE01/01510, filed Apr. 18, 2001 and designating the UnitedStates. This application further claims foreign priority to Germanapplication DE 100 20 125.3, filed Apr. 18, 2000.

This invention relates to an agent for postoperative use after removalof primary or metastatic bone tumors. This agent applies within themedical field, in particular in the field of orthopedics.

The prognosis for malignant bone tumors has undergone a definite changein the last two decades. In 1970 the five-year survival rate was lessthan 20%, but today almost 80% of patients survive. This success isattributed to recent therapeutic approaches with pre- or postoperativechemotherapy and/or radiation as well as expansion of the diagnostic andsurgical options, which permit a differentiated surgical procedureaccording to entity, tumor extent and grading. The goal of surgicaltreatment, apart from a few exceptions, is complete removal of thetumor. There are various options for the procedure in removing a tumor:

-   (1) preserving the extremity in the original shape, bridging any    resulting bone defect (limb salvage),-   (2) segment amputation,-   (3) amputation,    but method (1) is preferred in terms of the patient's quality of    life. The following table shows the different resection limits with    their pathological results:

Resection limits and pathological evaluation Resection levelPathological result Intracapsular Intralesional Resection margin in thetumor Marginal Extracapsular, but Reactive tissue, in the accompanyingpossibly with satellite reactive tissue lesions of the tumor ExtensiveIn normal tissue, Tumor-free, possibly outside of reactive dislocatedmetastases tissue (2 to 3 cm) excised Radical ExtracompartmentalTumor-free resection margin

The risks entailed in surgery that salvages the extremity is apparentfrom the pathological findings, because individual tumor residues mayremain if resection is inadequate. These tumor residues can have anextremely negative effect on the patient's prognosis (T. Ozaki, A.Hillmann, N. Lindner, S. Blasius, W. Winkelmann: Chondrosarcoma of thepelvis, Clin. Orthop. 337, 1997, 226–239). In the case of malignantsarcomas of bone and soft tissue, essentially extensive or radicalresection is the goal (S. Toma, A. Venturino, G. Sogno, C. Formica, B.Bignotti, S. Bonassi, R. Palumbo: Metastatic bone tumors. Nonsurgicaltreatment. Outcome and survival, Clin. Orthop. 295, 1993, 246–251).Otherwise, a local recurrence rate of 60–90% must be assumed formarginal resections.

Pre- and/or postoperative treatment of bone tumors by chemotherapy orradiation therapy minimizes the recurrence problem. In addition to theknown side effects of this treatment, however, renewed surgicalprocedures are repeatedly required in a certain percentage of thesepatients. In addition, not all tumors respond identically to the sametreatment strategies. The high incidence of recurrences with a marginalresection in many cases makes an extensive or radical resection appearto be the preferred surgical method. However, the result of such surgeryis usually that reconstruction of the bone proves to be complicated.Various options are available for reconstruction of bone:

-   -   autologous reconstruction,    -   endoprosthetics and    -   allogenic implants.

Autologous reconstruction uses bone material from the patient, which isinserted in place of the bone removed. The possible removal of materialis limited here, so that only minor resections can be refilled again.Endoprosthetics consist of replacing the missing bone by prostheses ofbiocompatible materials. This bone substitute is to some extent verycomplicated and cost-intensive to manufacture. In treatment of youngpatients in particular, the problem arises that the prostheses do notadapt to the patient's growth, thus necessitating follow-up surgeries.Allogenic implants presuppose the existence of a functioning bone bank.There is a high incidence of complications in allogenic bone and jointreplacement in the traditional sense without a vascular connection. Thefracture rate over a period of years is more than 50% in the case ofdiaphysis implants of the lower extremities, and the infection rate isreported as approximately 10% to 30%.

More recent therapeutic approaches are based on implantation ofbiodegradable materials coated with recombinant growth factors (C. A.Kirker-Head, T. N. Gerhart, S. H. Schelling, G. E. Hennig, E. Wang, M.E. Holtrop: Long-term healing of bone using recombinant human bonemorphogenetic protein 2, Clin. Orthop. 318, 1995, 222–230). Preliminaryexperiments with recombinant growth factors have already been conductedon animal models (review article: E. H. Groeneveld and E. H. Burger:Bone morphogenetic proteins in human bone regeneration, Eur. J.Endocrinol. 142(1), 2000, 9–21). Methods of producing and using suchrecombinant growth factors are already known (U.S. Pat. No. 4,472,840,U.S. Pat. No. 4,563,489, U.S. Pat. No. 4,596,574, U.S. Pat. No.4,789,732, U.S. Pat. No. 4,795,804, U.S. Pat. No. 5,318,898, U.S. Pat.No. 5,393,739, U.S. Pat. No. 5,618,924, European Patent 0,409,472,German Patent 19 748 734). These growth factors serve the purpose ofimproving the healing of bone defects because they stimulate naturalbone growth. However, these factors have no effect on the persistenceand possible dissemination of any tumor cells that may still be presentpostoperatively.

It is also known that the proliferation of osteoblasts may be increasedand bone resorption of osteoclasts can be inhibited by a fragment of theHMW (high molecular weight) kininogen known as cysteine proteinaseinhibitor (U.S. Pat. No. 5,885,964). The use of this fragment isappropriate when osteogenesis by osteoblasts is diminished due to ageand bone resorption by osteoclasts is increased at the same time.

Furthermore, studies of the proteinases involved in tumor metastases andin bone resorption are known (C. Haeckel et al.: Proteinase expressionin dedifferentiated parosteal osteosarcoma, Arch. Pathol. Lab. Med. 123,1999, 213–221; K. Bjornland et al.: S1000A4 involvement in metastasis:deregulation of matrix metalloproteinases and tissue inhibitors ofmatrix metalloproteinases in osteosarcoma cells transfected with ananti-S100A4 ribozyme, Cancer Res. 59, 1999, 4702–4708). The mostimportant enzymes involved in this process are cysteine proteinases(cathepsins L, B), matrix metal proteinases (MMP-2, MMP-9) and theserine proteinase uPA. In addition, it is known that cathepsin K, acysteine proteinase of osteoclasts, is involved in bone resorption (P.Garnero et al.: The collagenolytic activity of cathepsin K is uniqueamong mammalian proteinases, J. Biol. Chem. 273, 1998, 32347–32352).

Growth factors of the TGF-β superfamily such as the BMPs (bonemorphogenetic proteins) are capable of inducing osteoneogenesis. Oneexample is BMP-2, which is described in the following articles: H. Itohet al.: Experimental spinal fusion with use of recombinant human bonemorphogenetic protein 2, Spine 24, 1999, 1402–1405; K. Yoshida et al.:Enhancement by recombinant human bone morphogenetic protein-2 of boneformation by means of porous hydroxyapatite in mandibular bone defects,J. Dent. Res. 78, 1999, 1505–1510. In addition, other BMPs have beendescribed by J. M. Wozney et al.: Novel regulators of bone formation:molecular clones and activities, Science 242, 1988, 1528–1534; S. Oidaet al.: Cloning and sequence of bone morphogenetic protein 4 (BMP-4)from a human placental cDNA library, DNA Seq. 5, 1995, 273–275; A. J.Celeste et al.: Identification of transforming growth factor-beta familymembers present in bone-inductive protein purified from bovine bone,Proc. Natl. Acad. Sci. USA 87, 1990, 9843–9847; E. Ozkaynak et al.: OP-1cDNA encodes an osteogenic protein in the TGF-beta family, EMBO J. 9,1990, 2085–2093; E. Ozkaynak et al.: Osteogenic protein-2, A new memberof the transforming growth factor-beta superfamily expressed early inembryogenesis, J. Biol. Chem. 267, 1992, 25220–25227; J. Hino et al.:cDNA cloning and genomic structure of human bone morphogeneticprotein-3B (BMP-3b), Biochem. Biophys. Res. Commun. 223, 1996, 304–310.

Endogenous proteinase inhibitors are also known. The sequence for humancystatin C was described by M. Abrahamson et al.: Molecular cloning andsequence analysis of cDNA coding for the precursor of the human cysteineproteinase inhibitor cystatin, C. FEBS Lett. 216, 1987, 229–233; thesequence of TIMP-2 was described by W. G. Stetler-Stevenson et al.:Tissue inhibitor of metalloproteinase-2 (TIMP-2) mRNA expression intumor cell lines and human tumor tissues, J. Biol. Chem. 265, 1990,13933–13938 and that of PAI-2 was described by R. D. Ye et al.: cDNAcloning and expression in Escherichia coli of a plasminogen activatorinhibitor from human placenta, J. Biol. Chem. 262, 1987, 3718–3725.

The object of this invention is to create an agent for postoperativeuse, i.e., after excision of primary or metastatic bone tumors, whichsupports successful bone regeneration and requires a less stressfulsurgery for the patient without the risk of a new tumor metastasis tothe treated bone. The patient's quality of life should be increasedthrough this type of bone resection with effective and long-lastingcontrol of tumor, whereby it is necessary to take into account not onlythe surgical procedure for bone resection per se but also theconsequences of the procedure.

According to this invention, an agent is prepared from a nucleic acid bylinking an essentially known sequence for bone growth promotion and aknown proteinase inhibitor by a variable spacer molecule, e.g., anoligonucleotide. This linkage results in a novel active ingredienthaving two functions.

When this bifunctional agent is used postoperatively after removal ofbone tumors, bone growth is supported and also metastasis (through tumorcells remaining in the surgical field) in the marginal zones of the boneprosthesis is inhibited.

Depending on the biological activity of the tumor, micro-metastases maybe expected in any case, leading to local relapses. In addition to thefavorable effect on reconstitution of the bone, the risk of a possiblemetastasis should be largely minimized. In contrast to practice, it isto be done without a radical resection which has the goal of removing asmuch bone material that the resection borders are reliably tumor-free inorder to effectively combat the tumor. Instead, only a minimum of bonematerial needs to be resected, whereby the risk of a further metastasisstarting from the resection margin is reduced. Because the procedure isminimal, the patient's quality of life is increased not only concerningthe surgery and its immediate consequences but also regarding laterconsequences. Due to the influence of the bifunctional agent, there isalso better growth into the prosthesis, which results in shorterrecovery times and more stable incorporation of the prosthesis, amongother effects. This should also prevent follow-up surgeries.

A DNA according to the invention is described below in the form of acDNA. This stands exemplarily forany DNA falling under the presentinvention. The agent is further described as a bifunctional protein andis produced with the help of well-known methods of genetic engineering.The basis of the bifunctional protein may be two independently naturallyoccurring proteins or domains of proteins, which are linked with oneanother by means of a spacer molecule (a peptide not belonging to thenatural protein domains between the functional domains).

This invention includes the coupling of two cDNAs by an oligonucleotideto form a new cDNA. This invention also includes derivatives of this newcDNA, which are formed by replacement, insertion, or deletion of one ormore nucleotides, where the activity of the coded gene product ispreserved. An object of this invention is also recombinant expression ofthe bifunctional protein in prokaryotes such as E. coli strains. This isdone by using vectors, which permit expression in prokaryotic cells.These vectors contain suitable well-known regulation signals for geneexpression such as promoters and ribosome binding sites. The promotorsused include, for example, the T7 promotor, the tac promotor and the tetpromotor. The vectors also code for antibiotic resistence and thereplication origin.

The cDNA of the bifunctional protein is used for in vitro and in vivotransfection of suitable cell cultures such as cells of mesenchymalorigin. Transfection is understood to refer to the insertion of nucleicacid constructs into cells or tissue. To do so, vectors containingwell-known and suitable regulation signals for gene expression are used.These include transcription signals such as promoters, enhancers, andpolyadenylation sites as well as translation signals such as ribosomebinding sites. Promotors used include eukaryotic promoters of viral andcellular origin such as the CMV promotor, the RSV promotor or the -actinpromotor. All known polyadenylation signals may be used as thepolyadenylation signal, e.g., that of SV40. These vectors mayadditionally contain genetic markers such as antibiotic resistancegenes. Furthermore, viral vectors are also suitable for transfection ofcells.

DNA constructs for prokaryotic and eukaryotic expression are produced bythe well-known methods of genetic engineering such as PCR and cloning.

This invention will now be explained in greater detail on the basis ofexemplary embodiments illustrated in the drawings, which show:

FIG. 1 primer used in PCR FIG. 2 expression plasmid for the fusionprotein of cystatin C and BMP-2 FIG. 3 examples of possibleoligonucleotides for linking the fusion partners FIG. 4 expressionplasmid for the fusion protein of cystatin C and BMP-2 in a plasmid withmaltose binding protein (MBP)

Embodiment 1:

Producing the Prokaryotic Expression Plasmid

The cDNA of human cystatin C was amplified by means of primers A and Bshown in FIG. 1 for the polymerase chain reaction (PCR) from a plasmid(M. Abrahamson et al.: Molecular cloning and sequence analysis of cDNAcoding for the precursor of the human cysteine proteinase inhibitorcystatin C, FEBS Lett. 216, 1987, 229–233). The cDNA of a mature humanBMP-2 was obtained from the plasmid pBF008 (B. Fahnert, HKI Jena) byrestriction digestion with the restriction endonucleases Eco47III andHindIII. Both cDNAs were cloned consecutively in reading frame in theexpression plasmid pASK75 (Biometra). For this purpose firstly the PCRproduct of human cystatin C was cloned in the vector pCR2.1-TOPO(Invitrogen) and then the correctness of the PCR product was checkedwith the LICOR system. The cDNA of human cystatin C was cut out byrestriction digestion with the enzymes XbaI and Eco47III and was ligatedinto the vector pASK75, which was cleaved with the same restrictionenzymes. The resulting construct (pASKcC) was digested with therestriction enzymes Eco47III and HindIII. The cDNA of the mature humanBMP-2 cut out of the plasmid pBF008 with the same restriction enzyme wasligated into the previously cleaved plasmid pASK75cC. A peptideconsisting of six histidines (SEQ ID NO. 10), located on the N-terminusof the cDNA of mature human BMP-2, was used as a possible spacer betweenthe two cDNAs in the cloning strategy described here (see also SEQ IDNOS. 12, 14, 16, 18). The base sequence in the regions of the DNAconstruct that were of interest was checked by DNA sequencing using theLICOR system, and it was verified that the reading frame is correct.FIG. 2 shows the resulting expression plasmid (pASKcCHBMP-2). FIG. 3shows examples of possible oligonucleotides for linking the fusionpartners. Exemplarily cystatin C is mentioned as an inhibitor and BMP-2as a growth factor. For example, PAI-2 could also be used as aproteinase inhibitor for serine proteinases, or TIMP-2 could be used asan inhibitor for metalloproteinases. Other growth factors that could beused include BMP-3, BMP-4, BMP-7 and other representatives of the TGF-βsuperfamily.

The cDNA of the bifunctional protein (cCHBMP-2) was amplified by PCRwith primers C, D and optionally D′ (FIG. 1) and inserted in the plasmidpMALc2 (New England BioLabs). For this purpose the PCR product cCHBMP-2was cloned in the vector pCR2.1-TOPO and was cut out with therestriction enzymes BamHI and HindIII. The plasmid pMALc2 was digestedwith the same restriction enzymes. The two cDNAs were ligated together.The resulting expression plasmid was sequenced in the region ofinterest. This expression plasmid (pMALc2cCHBMP-2, FIG. 4) was used forexpression of the bifunctional protein as a fusion protein with anotherprotein, the protein MBP (maltose binding protein). MBP is used forpurification of recombinant proteins by affinity chromatography. It canbe removed again by factor Xa cleavage after purification, so that theauthentic N-terminus of cystatin C is preserved.

Embodiment 2:

Expression of the Bifunctional Protein in E. coli (BL21(DE3))

For the purpose of expression, the expression plasmids pASKcCHBMP-2(FIG. 2) and pMALc2cCHBMP-2 (FIG. 4), which are described in Embodiment1, were transformed in the bacterial strain BL21 (DE3) (Novagen). Thetransformation was performed by well-known methods for chemicallycompetent cells.

Expression was performed as follows. 200 mL Terrific broth (TB) mediumwith 100 g/mL ampicillin was inoculated with a single clone andcultured. Expression was induced by adding 100 g/L anhydrotetracyclinein the case of plasmid paskcCHBMP-2 and by adding 1 mM IPTG in the caseof plasmid pMalc2cCHBMP-2.

The bacteria were harvested by centrifugation and disrupted bywell-known methods with lysis buffer and sonication. This material waspurified by chromatography on Ni-NTA resin under denaturing conditions.

The folding and dimerization were performed in a dimerization buffer at25° C. over a period of several days.

In addition to expression in bacteria, which is presented here as anexample, expression in other known expression systems such as yeasts,insect cells or mammalian cells is also possible.

1. An isolated nucleic acid encoding a bifunctional protein comprising:(a) an isolated nucleic acid encoding bone growth factor BMP-2 capableof inducing osteogenesis; (b) an isolated nucleic acid encoding aproteinase inhibitor, wherein said nucleic acid of (a) is linked to saidnucleic acid of (b) via an oligonucleotide, and wherein said so linkednucleic acids encode a bifunctional protein.
 2. The nucleic acidencoding a bifunctional protein of claim 1, wherein said nucleic acidencoding the proteinase inhibitor is a sequence encoding a cysteineproteinase inhibitor or a functional fragment thereof and wherein saidfunctional fragment retains the function of said proteinase inhibitorencoded by said nucleic acid of (b).
 3. The nucleic acid encoding abifunctional protein of claim 1, wherein said nucleic acid encoding theproteinase inhibitor is a sequence encoding a serine proteinaseinhibitor or a functional fragment thereof and wherein said functionalfragment retains the function of said proteinase inhibitor encoded bysaid nucleic acid of (b).
 4. The nucleic acid encoding a bifunctionalprotein of claim 1, wherein said nucleic acid encoding the proteinaseinhibitor is a sequence encoding a metalloproteinase inhibitor or afunctional fragment thereof and wherein said functional fragment retainsthe function of said proteinase inhibitor encoded by said nucleic acidof (b).
 5. The nucleic acid encoding a bifunctional protein of claim 1,wherein the oligonucleotide linking (a) and (b) is SEQ ID NO: 10 or afunctional fragment thereof.
 6. The nucleic acid encoding a bifunctionalprotein of claim 1, wherein the oligonucleotide linking (a) and (b) ischosen from the group consisting of SEQ ID NO: 12, 14, 16, 18, or afunctional fragment thereof.
 7. A method for producing a bifunctionalprotein, said method comprising: (a) providing an isolated nucleic acidencoding BMP-2 capable of inducing osteogenesis, (b) linking saidisolated nucleic acid to another isolated nucleic acid encoding aproteinase inhibitor via an oligonucleotide, and expressing saidbifunctional protein in vitro, wherein said bifunctional protein retainsthe functions of said bone growth factor BMP-2 and said proteinaseinhibitor.
 8. The method of claim 7, wherein said nucleic acid encodingthe proteinase inhibitor is a sequence encoding a cysteine proteinaseinhibitor or a functional fragment thereof and wherein said functionalfragment retains the function of said proteinase inhibitor encoded bysaid nucleic acid of (b).
 9. The method of claim 7, wherein said nucleicacid encoding the proteinase inhibitor is a sequence encoding a serineproteinase inhibitor or a functional fragment thereof and wherein saidfunctional fragment retains the function of said proteinase inhibitorencoded by said nucleic acid of (b).
 10. The method of claim 7, whereinsaid nucleic acid encoding the proteinase inhibitor is a sequenceencoding a metalloproteinase inhibitor or a functional fragment thereofand wherein said functional fragment retains the function of saidproteinase inhibitor encoded by said nucleic acid of (b).
 11. The methodof claim 7, wherein the oligonucleotide linking the BMP-2 and proteinaseinhibitor encoding sequences is SEQ ID NO: 10 or a functional fragmentthereof.
 12. The method of claim 7, wherein the oligonucleotide linkingthe BMP-2 and proteinase inhibitor encoding sequences is chosen from thegroup consisting of SEQ ID NO: 12, 14, 16, 18, or a functional fragmentthereof.
 13. A method for producing a nucleic acid encoding abifunctional protein, said method comprising (a) providing an isolatednucleic acid encoding a BMP-2 capable of inducing osteogenesis, (b)linking said isolated nucleic acid to another isolated nucleic acidencoding a proteinase inhibitor via an oligonucleotide, wherein the solinked nucleic acids encode said bifunctional protein, wherein saidbifunctional protein retains the two functions of said BMP-2 and saidproteinase inhibitor encoded by said nucleic acids of (a) and (b). 14.The method of claim 13, wherein the nucleic acid encoding the proteinaseinhibitor is a sequence encoding a cysteine proteinase inhibitor or afunctional fragment thereof.
 15. The method of claim 13, wherein thenucleic acid encoding the proteinase inhibitor is a sequence encoding aserine proteinase inhibitor or a functional fragment thereof.
 16. Themethod of claim 13, wherein the nucleic acid encoding the proteinaseinhibitor is a sequence encoding a metalloproteinase inhibitor or afunctional fragment thereof.
 17. The method of claim 13, wherein theoligonucleotide is encoded by SEQ ID NO: 10 or a functional fragmentthereof.
 18. The method of claim 13, wherein the oligonucleotide ischosen from the group consisting of SEQ ID NO: 12, 14, 16, 18, or afunctional fragment thereof.